Article Figures & Data

Figures

A, structure of 4-HPR. B, 4-HPR enhances apoptosis induced by TNF and chemotherapeutic agents. KBM-5 cells (5,000/0.1 mL) were incubated at 37°C with TNF, Taxol, or doxorubicin (Doxo) in the presence and absence of 5 μmol/L 4-HPR, as indicated, for 72-hour duration, and the viable cells were assayed using MTT reagent. Columns, mean cytotoxicity from triplicate cultures; bars, SD. C, 4-HPR enhances TNF-induced PARP cleavage. KBM-5 cells (2 × 106/1 mL) were serum starved for 24 hours and then incubated with TNF (1 nmol/L) alone or in combination with 4-HPR (25 μmol/L) for the indicated times, and PARP cleavage was determined by Western blot analysis as described in Materials and Methods. Values (bottom) indicate the densitometry analysis of the 87,000 Da band. D, 4-HPR enhances TNF-induced cell death. KBM-5 cells (2 × 106/mL) were serum starved for 24 hours and then incubated with TNF (1 nmol/L) alone or in combination with 4-HPR (10 μmol/L) as indicated for 24 hours. Cell death was determined by calcein-AM–based Live and Dead assay as described in Materials and Methods. Representative experiment of the three independent ones showing similar results.

4-HPR suppresses TNF-induced invasive activity and RANKL-induced osteoclastogenesis. A, H1299 cells (2.5 × 104) were seeded into the upper wells of a Matrigel invasion chamber overnight in the absence of serum, pretreated with 10 μmol/L 4-HPR for 12 hours, treated with 1 nmol/L TNF for 24 hours in the presence of 1% serum, and then subjected to invasion assay. The value for no 4-HPR and no TNF was set to 1.0. Columns, mean of three experiments; bars, SD. *, P < 0.05; **, P < 0.001. B, RAW 264.7 cells (1 × 104) were plated overnight, pretreated with 5 μmol/L 4-HPR for 12 hours, and then treated with 5 nmol/L RANKL. Four and 5 days later, cells were stained for TRAP and evaluated for osteoclastogenesis. Photographs were taken after 5 days of incubation with RANKL. The numbers of TRAP-positive multinucleated osteoclasts (>3 nuclei) per well were counted (C).

4-HPR inhibits TNF-induced NF-κB–regulated gene products. A, 4-HPR inhibits MMP-9, COX-2, and VEGF expression induced by TNF. H1299 cells (2 × 106/mL) were left untreated or incubated with 25 μmol/L 4-HPR for 24 hours and then treated with 0.1 nmol/L TNF for different times. Whole-cell extracts were prepared, and whole-cell lysate (80 μg) was analyzed by Western blotting using antibodies against VEGF, MMP-9, and COX-2. B, 4-HPR inhibits cyclin D1 and c-myc expression induced by TNF. H1299 cells (2 × 106/mL) were left untreated or incubated with 25 μmol/L 4-HPR for 24 hours and then treated with 0.1 nmol/L TNF for different times. Whole-cell extracts were prepared, and whole-cell lysate (80 μg) was analyzed by Western blotting using antibodies against cyclin D1 and c-myc. Representative experiment of the three independent ones showing similar results. C, 4-HPR inhibits the expression of antiapoptotic gene products cIAP1, Bfl-1/A1, Bcl-2, TRAF1, and cFLIP: H1299 cells (2 × 106/mL) were left untreated or incubated with 25 μmol/L 4-HPR for 24 hours and then treated with 0.1 nmol/L TNF for different times. Whole-cell extracts were prepared and whole-cell lysate (50 μg) was analyzed by Western blotting using antibodies against IAP1, Bfl-1/A1, Bcl-2, TRAF1, and cFLIP as indicated. Representative experiment of the three independent ones showing similar results.

A, 4-HPR inhibits TNF-induced phosphorylation and degradation of IκBα. H1299 cells (2 × 106/mL) were incubated with 25 μmol/L 4-HPR for 24 hours at 37°C, treated with 0.1 nmol/L TNF for the indicated times at 37°C, and then tested for IκBα (top) and phosphorylated IκBα (middle) in cytosolic fractions by Western blot analysis. Equal protein loading was evaluated by β-actin (bottom). B, 4-HPR inhibits TNF-induced IKK activity. H1299 cells (2 × 106/mL) were treated with 25 μmol/L 4-HPR for 24 hours and then treated with 0.1 nmol/L TNF for the indicated time intervals. Whole-cell extracts were prepared and extract (200 μg) was immunoprecipitated with antibodies against IKKα and IKKβ. Thereafter, immune complex kinase assay was done as described in Materials and Methods. To examine the effect of 4-HPR on the level of expression of IKK proteins, whole-cell extract (30 μg) was run on 10% SDS-PAGE, electrotransferred, and immunoblotted with indicated antibodies as described in Materials and Methods. C, 4-HPR has no direct effect on IKK activity. Whole-cell extracts were prepared from untreated and TNF (0.1 nmol/L)–treated H1299 cells (2 × 106/mL); protein/sample whole-cell extract (200 μg) was immunoprecipitated with antibodies against IKKα and IKKβ. The immune complex was treated with the indicated concentrations of 4-HPR for 30 minutes at 30°C; then, a kinase assay was done as described in Materials and Methods. Equal protein loading was evaluated by IKKβ. D, 4-HPR inhibits Akt phosphorylation. H1299 cells (2 × 106/mL) were incubated with 25 μmol/L 4-HPR for 24 hours and then treated with 0.1 nmol/L TNF for the indicated times. The whole cell extracts were analyzed by Western blotting using antibodies against the phosphorylated Akt. Equal protein loading was evaluated by Akt. Representative experiment of the three independent ones showing similar results. E, 4-HPR inhibits TNF-induced phosphorylation of p65. H1299 cells (2 × 106/mL) were incubated with 25 μmol/L 4-HPR for 24 hours and then treated with 0.1 nmol/L TNF for the indicated times. The cytoplasmic extracts were analyzed by Western blotting using antibodies against the phosphorylated form of p65. F, 4-HPR inhibits TNF-induced nuclear translocation of p65. H1299 cells (1 × 106/mL) were either untreated or pretreated with 25 μmol/L 4-HPR for 24 hours at 37°C and then treated with 0.1 nmol/L TNF for the indicated times. Nuclear extracts were prepared and analyzed by Western blotting using antibodies against p65. G, 4-HPR inhibits TNF-induced nuclear translocation of p65. H1299 cells (1 × 106/mL) were first treated with 25 μmol/L 4-HPR for 24 hours at 37°C and then exposed to 0.1 nmol/L TNF. After cytospin, immunocytochemical analysis was done as described in Materials and Methods. Representative experiment of the three independent ones showing similar results.

A, 4-HPR inhibits TNF-induced NF-κB–dependent reporter gene (SEAP) expression. H1299 cells were transiently transfected with a NF-κB-containing plasmid linked to the SEAP gene and then treated with the indicated concentrations of 4-HPR. After 24 hours in culture with 0.1 nmol/L TNF, cell supernatants were collected and assayed for SEAP activity as described in Materials and Methods. Results are expressed as fold activity over the activity of the vector control. B, 4-HPR inhibits NF-κB–dependent reporter gene expression induced by TNFR, TRADD, TRAF, NIK, and IKKβ. H1299 cells were transiently transfected with the indicated plasmids along with a NF-κB-containing plasmid linked to the SEAP gene and then left either untreated or treated with 25 μmol/L 4-HPR for 24 hours. Cell supernatants were assayed for SEAP activity as described in Materials and Methods. Results are expressed as fold activity over the activity of the vector control. Bars, SD. C, 4-HPR inhibits TNF-induced COX-2 promoter activity. H1299 cells were transiently transfected with a COX-2 promoter plasmid linked to the luciferase gene and then treated with the indicated concentrations of 4-HPR. After 24 hours in culture with 0.1 nmol/L TNF, cell supernatants were collected and assayed for luciferase activity as described in Materials and Methods. Results are expressed as fold activity over the activity of the vector control. Columns, mean of triplicate cultures of a representative experiment of the three independent ones showing similar results; bars, SD. D, schematic representation of the effect of 4-HPR on TNF-induced NF-κB activation and apoptosis.